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Cloning and heterologous expression of P450Lent4B11, a novel bacterial P450 gene, for hydroxylation of an antifungal agent sordaricin.
The Journal of Antibiotics ( IF 3.3 ) Pub Date : 2020-05-01 , DOI: 10.1038/s41429-020-0310-9
Motoi Ueno 1 , Motoo Kobayashi 2 , Akihiko Fujie 3 , Takashi Shibata 4
Affiliation  

Microbial transformation is known to be one of promising options to add functional groups such as a hydroxyl moiety to active base compounds to generate their derivatives. Sordaricin, a diterpene aglycone of the natural product sordarin, is an antifungal agent to selectively inhibit fungal protein synthesis by stabilizing the ribosome/EF-2 (elongation factor 2) complex. We screened actinomycetes to catalyze hydroxylation of sordaricin on the basis that the hydroxyl moiety would make it easier to generate derivatives of sordaricin. As a result of the screening, 6-hydroxylation of sordaricin was found to be catalyzed by Lentzea sp. 7887. We found that the cytochrome P450 inhibitor metyrapone inhibited this reaction, suggesting that a cytochrome P450 may be responsible for the biotransformation. As a next step, we cloned multiple cytochrome P450 genes, one of which were named P450Lent4B11, using degenerate PCR primers. The expressed cytochrome P450 derived from the P450Lent4B11 gene provided a different absorbance spectrum pattern from original one when it was incubated with sordaricin. Moreover, in cell-free conditions, the corresponding cytochrome P450 displayed the 6-hydroxylation activity toward sordaricin. Taken together, these results indicate that P450Lent4B11, derived from Lentzea sp. 7887, should be responsible for catalyzing 6-hydroxylation of sordaricin.

中文翻译:

一种新型细菌P450基因P450Lent4B11的克隆和异源表达,用于抗真菌药索达霉素的羟基化。

已知微生物转化是将诸如羟基部分的官能团添加至活性碱化合物以生成其衍生物的有前途的选择之一。索达霉素是天然产物索达林的二萜糖苷配基,是一种通过稳定核糖体/ EF-2(伸长因子2)复合物来选择性抑制真菌蛋白质合成的抗真菌剂。我们筛选了放线菌以催化苏打霉素的羟基化,其基础是羟基部分将使其更易于生成苏打霉素的衍生物。筛选的结果表明,Lendtzea sp。催化了苏达霉素的6-羟基化。7887.我们发现细胞色素P450抑制剂美拉普酮抑制了该反应,这表明细胞色素P450可能是生物转化的原因。下一步,我们使用简并PCR引物克隆了多个细胞色素P450基因,其中一个名为P450Lent4B11。当与索达霉素一起孵育时,表达自P450Lent4B11基因的细胞色素P450提供了不同于原始细胞色素的吸收光谱模式。此外,在无细胞条件下,相应的细胞色素P450对苏打霉素具有6-羟基化活性。综上所述,这些结果表明衍生自Lentzea sp。的P450Lent4B11。7887,应负责催化索达霉素的6-羟基化反应。相应的细胞色素P450表现出对索达霉素的6-羟基化活性。综上所述,这些结果表明衍生自Lentzea sp。的P450Lent4B11。7887,应负责催化索达霉素的6-羟基化反应。相应的细胞色素P450表现出对索达霉素的6-羟基化活性。综上所述,这些结果表明衍生自Lentzea sp。的P450Lent4B11。7887,应负责催化索达霉素的6-羟基化反应。
更新日期:2020-05-01
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